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1 DOI: /ncb2419 Figure S1 NiGFP localization in Dl mutant dividing SOPs. a-c) time-lapse analysis of NiGFP (green) in Dl mutant SOPs (H2B-RFP, red; clones were identified by the loss of nlsgfp) showing three dividing mutant SOPs, colorcoded with dots at t=0 (a) and t=10 mn (b-c ). NiGFP specifically accumulated at the apical interface between daughter cells (surface view in c). NiGFP was not detected in the nuclei of Dl SOP progeny cells (white dots in c ). Genotype: N 55e11 w 1118 P[Ubx-flp] P[neur-Histone2B-RFP]619 / Y ; M[3xP3-RFP.attP. w+.nigfp]51d / + ; FRT82B P[ubi-nlsGFP] / FRT82B Dl Rev
2 Figure S2 Cell fate and nuclear NiGFP in numb RNAi and spdo RNAi pupae. a-e) the levels of nuclear NiGFP (green) were examined in SOPs (marked by H2B-RFP, red) in living pupae of the following genotypes: wild-type (i.e. N 55e11 NiGFP; a-a ), numb RNAi (N 55e11 NiGFP numb RNAi ; b-b,d) and spdo RNAi (N 55e11 NiGFP spdo RNAi ; c-c, e) pupae. Panels a-c show projected views of 3 confocal sections (with a Dz of 0.24mm; NiGFP channels are shown in a, b and c ). The corresponding intensity profile are shown in a, b and c (H2B-RFP profile in red and NiGFP profile in green). Nuclear NiGFP levels appeared to be higher in piia than in piib in wild-type controls (a ). Similarly, nuclear NiGFP levels appeared to be higher in numb RNAi piia and piia-like cells (b ) than in spdo RNAi piib and piib-like cells (c ) relative to their epidermal neighbours. Nuclear NiGFP was monitored over time as in Fig. 2e: no difference in nuclear NiGFP was detected between daughter cells upon silencing of numb (d; red line) and spdo (e; red line). The wild-type control (green line) shown in Fig. 2e is shown here as a reference. f-i) Analysis of cell fate transformations upon the silencing of spdo and/or numb. The fate of the sensory cells was examined in wild-type (f), numb RNAi (g), spdo RNAi (h) and numb RNAi spdo RNAi (i) pupae at hr APF using Cut (a nuclear sensory organ cell marker, red), HRP (a neuron marker, blue) and Su(H) (a socket cell marker, green). Silencing of numb phenocopied the numb mutant phenotype and led to piib-topiia transformations, as revealed by the loss of HRP-positive neurons and the gain of Su(H)-positive socket cells. The silencing of spdo also phenocopied the spdo mutant phenotype and resulted in opposite piia-topiib transformations, as revealed by the loss of Su(H)-positive socket cells and the gain of HRP-positive neurons. Double silencing of numb and spdo had the same effect as the silencing of spdo alone, consistent with spdo being epistatic over numb. Genotypes: a-a : N 55e11 w 1118 P[neur-Histone2B-RFP]619 / Y ; M[3xP3-RFP.attP. w+.nigfp]51d / + b-b, d: N 55e11 w 1118 P[neur-Histone2B-RFP]619 / Y ; M[3xP3-RFP.attP. w+.nigfp]51d / + ; pnr-gal4 P[UAS-dsRNA numb]-3779r-3 / + c-c, e: N 55e11 w 1118 P[neur-Histone2B-RFP]619 / Y ; M[3xP3-RFP.attP. w+.nigfp]51d / P[UAS-dsRNA spdo] ; pnr-gal4 / + f: w 1118 g: w 1118 ; ; pnr-gal4 P[UAS-dsRNA numb]-3779r-3 / + h: w 1118 P[UAS-dsRNA spdo] / + ; pnr-gal4 / + i: w 1118 P[UAS-dsRNA spdo] / + ; pnr-gal4 P[UAS-dsRNA numb]- 3779R-3 / + 2
3 Figure S3 Live analysis of NiGFP localization upon conditional inhibition of endocytosis. NiGFP (green) localisation in shi ts pupae. At permissive temperature (22 C; t=-1), NiGFP localized in relatively uniform manner at the cell cortex of epidermal cells (top panels: surface views at z=0mm) and in small apical endosomes (middle panels, z=-1 mm) in shi ts pupae. Temperature was shifted at restrictive temperature (32 C) at t=0 (telophase was monitored in SOPs using H2B-RFP, red). NiGFP progressively accumulated into distinct foci at the apical cortex (surface views) and NiGFP containing endosomes disappeared (z=-1 mm) and NiGFP accumulated in dots along the piia/piib interface 3-5 mm below the apical surface (bottom panels). These changes were not observed in shi ts pupae at 22 C, nor in wild-type pupae at 32 C (not shown; see Fig. 4d-d ). Genotype: shi ts / Y ; M[3xP3-RFP.attP.w+.NiGFP]51D / + ; P[neur-Histone2B-RFP] / + 3
4 Figure S4 numb-independent inhibition of Notch by Spdo. a-b ) Ectopic expression of Spdo in MARCM clones (GFP, green) resulted in increased SOP density (Senseless, red) in developing nota at 16 hr APF. This increased density is indicative of reduced Notch signalling. This inhibitory effect of Spdo on Notch activity was seen in both wild-type cells (a,a ) and numb mutant cells (b,b ), indicating that Spdo overexpression can inhibit the activity of Notch even in the absence of Numb. c) Analysis of cell fate decisions along clone borders in developing nota at 16 hr APF (as shown in a-b ). The bars illustrate the percentage of GFP-negative (red; outside the clone) and GFP-positive (yellow; inside the clone) SOPs that are located along clone borders (% values, given on the y-axis, are indicated above each bar; n is the total number of SOPs scored along clone borders). The genotypes scored in this clone border analysis are indicated above each pair of red/ yellow bars. GFP positive cells are either mutant and/or expressing spdo. Cells expressing Spdo prior to SOP selection were found to be more likely to become SOPs than their wild-type neighbors. This indicates that cells with high Spdo activity have low Notch receptor activity 1. This inhibitory effect of Spdo overexpression on Notch was also observed in numb mutant clones, showing that Spdo can down-regulate Notch in a Numb-independent manner. Since spdo cells mutant have the same probability to become SOPs than their wild-type neighbors, we conclude that endogenous Spdo has no significant role in the process of SOP selection. Control wild-type clones were generated using the parental FRT40A and FRT82B chromosomes. Genotypes: a, c: w 1118 hs-flp ptub-gal4 UAS-nlsGFP ; FRT40A / FRT40A ptub-gal80 ; P[UAS-Spdo]G1 / + b, c: w 1118 hs-flp ptub-gal4 UAS-nlsGFP ; FRT40A numb 15 / FRT40A ptub-gal80 ; P[UAS-Spdo]G1 / + c: w 1118 hs-flp ptub-gal4 UAS-nlsGFP ; FRT40A / FRT40A ptub-gal80 c: w 1118 hs-flp ptub-gal4 UAS-nlsGFP ; FRT40A numb 15 / FRT40A ptub- GAL80 c: w 1118 hs-flp ptub-gal4 UAS-nlsGFP ; FRT82B spdo G104 / FRT82B ptub- GAL80 c: w 1118 hs-flp ptub-gal4 UAS-nlsGFP ; FRT82B / FRT82B ptub-gal80 4
5 Figure S5 endocytosis of Notch by Spdo did not strictly require the NPAF motif of Spdo. Expression of GFP-Spdo (green in a-c ) and GFP-Spdo NP>AA (green in d-f ; the NPAF motif of Spdo that mediates interaction with Numb is deleted in GFP-Spdo NP>AA 2 ) in wing imaginal cells (ectopic expression of Spdo was directed in dorsal cells using ap-gal4) resulted in reduced levels of Notch at the apical cortex (NECD, red). Note that GFP-Spdo NP>AA localized baso-laterally whereas GFP-Spdo localized more apically. Panels b, b, e and e show high magnification views of a and d, respectively. Cross section views are shown in panels c, c, f and f. We conclude that the Spdo- Numb interaction is not strictly required for the down-regulation of Notch by Spdo. Genotypes: a-c : w 1118 ; ap-gal4 / UAS-GFP-Spdo d-f : w 1118 ; ap-gal4 / + ; UAS-GFP-Spdo NP>AA / + 5
6 Figure S6 Numb enhanced the down-regulation of Notch by Spdo. Analysis of the density of SOPs (marked by Senseless) in 16 hr APF pupae was used to investigate the effect of Numb and Spdo on the activity of Notch. Mild overexpression of Numb (b) and Spdo (c) had a weak inhibitory effect on Notch (compare with the negative control in a), as revealed by a mild increase in SOP number. Concomitant overexpression of Numb and Spdo resulted in a significant increase in SOP density (d), indicating that Numb enhanced the inhibitory effect of Spdo on the activity of Notch. Genotypes: a: w 1118 ; ; eq-gal4 / + b: w 1118 ; ; eq-gal4 / P[UAS-Numb] c: w 1118 ; ; eq-gal4 / P[UAS-Spdo]G1 d: w 1118 ; ; eq-gal4 / P[UAS-Numb] P[UAS-Spdo]G1 6
7 Supplemental Information - Movies Movie 1: live imaging of Notch in a wild-type SOP Spinning disk confocal sections corresponding to surface (z=0), subapical (z=-1mm) and basal (z=-5mm) views of NiGFP (green/white) are shown together with H2B-RFP (magenta; maximal projection of several confocal sections). Genotype: N 55e11 w 1118 P[neur-Histone2B-RFP]619 / Y ; M[3xP3-RFP.attP.w+.NiGFP]51D / + Movie 2: live imaging of cytokinesis in a wild-type SOP Actin (sgmca, white) and Histone2B (H2B-RFP, red) were imaged using spinning disk confocal microscopy. Confocal sections corresponding to surface (z=0), subapical (z=-3mm) and basal (z=-5mm) views are shown. A maximal projection of H2B-RFP channel is shown. Cytokinetic furrow initially forms basally. Furrow ingression is concomitant to chromatin decondensation. Note that apical actin accumulates in the newly born SOP daughter cells. This accumulation has been described as the Actin Rich Structure (ARS) 3. Genotype: w 1118 P[pNeur-H2B-RFP]619 / Y; P[pSqh-MoeABD-GFP] (sgmca ) /+ Movie 3: live imaging of Notch in a numb RNAi SOP Spinning disk confocal sections corresponding to surface (z=0) and basal (z=-5mm) views of NiGFP (green/white) are shown together with H2B-RFP (magenta; maximal projection of several confocal sections). A transient basal accumulation of NiGFP is indicated by an arrow. Genotype: N 55e11 w 1118 P[neur-Histone2B-RFP]619; M[3xP3-RFP.attP.w+.NiGFP]51D / + ; pnr-gal4 P[UAS-dsRNA numb]-3779r-3 / + Movie 4: live imaging of Notch in a spdo RNAi SOP Spinning disk confocal sections corresponding to surface (z=0) and subapical (z=-1mm) views of NiGFP (green/white) are shown together with H2B-RFP (magenta; maximal projection of several confocal sections). A transient apical accumulation of NiGFP is indicated by an arrow. Genotype: N 55e11 w 1118 P[neur-Histone2B-RFP]619; M[3xP3-RFP.attP.w+.NiGFP]51D / P[UAS-dsRNA spdo] ; pnr-gal4 / + 7
8 GENOTYPES PANELS w 1118 shi ts1 N 55e11 w 1118 / Y ; M[3xP3-RFP.attP.w+.NiGFP]51D / + N 55e11 w 1118 P[neur-Histone2B-RFP]619 / Y ; M[3xP3- RFP.attP.w+.NiGFP]51D / + N 55e11 w 1118 P[neur-PH-mRFP1]-965 / Y ; P[neur-PHmRFP1]-967 M[3xP3-RFP.attP.w+.NiGFP]51D / + d-d : N 55e11 w 1118 P[Ubx-flp] P[neur-Histone2B- RFP]619 / Y ; M[3xP3-RFP.attP.w+.NiGFP]51D / + ; FRT82B P[ubi-nlsGFP] / FRT82B neur IF65 N 55e11 w 1118 P[neur-PH-mRFP1]-965 / Y ; P[neur-PHmRFP1]-967 M[3xP3-RFP.attP.w+.NiGFP]51D / + N 55e11 w 1118 P[neur-Histone2B-RFP]619 / Y ; M[3xP3- RFP.attP.w+.NiGFP]51D / + ; pnr-gal4 P[UAS-dsRNA numb]-3779r-3 / + N 55e11 w 1118 P[neur-Histone2B-RFP]619 / Y ; M[3xP3- RFP.attP.w+.NiGFP]51D / P[UAS-dsRNA spdo] ; pnr-gal4 / + N 55e11 w 1118 P[neur-Histone2B-RFP]619 / Y ; M[3xP3- RFP.attP.w+.NiGFP]51D / P[UAS-dsRNA spdo] ; pnr-gal4 P[UAS-dsRNA numb]-3779r-3 / + N 55e11 w 1118 P[Ubx-flp] P[neur-Histone2B-RFP]619 / Y ; numb 15 FRT40A M[3xP3-RFP.attP.w+.NiGFP]51D / P[ubi-nlsGFP] FRT40A N 55e11 w 1118 P[Ubx-flp] P[neur-Histone2B-RFP]619 / Y ; M[3xP3-RFP.attP.w+.NiGFP]51D / + ; FRT82B P[ubinlsGFP] / FRT82B spdo G104 Fig. 2b,b ; Fig. 4b,b ; Fig. 4f; Fig. 6d-d Fig. 4c,c Fig. 2a,a Fig. 1c-c ; Fig. 1e-g ; Fig. 2c,c ; Fig. 2e; Fig. 3a; Fig. 4d-d ; Fig. 5a Fig. 2d Fig. 1d-d Fig. 1i,i' Fig. 3b; Fig. 4g Fig. 3c; Fig. 4h; Fig. 5b Fig. 3d Fig. 3e Fig. 3f
9 N 55e11 w 1118 P[neur-PH-mRFP1]-965 / Y ; M[3xP3- RFP.attP.w+.NiGFP]51D / + ; pnr-gal4 P[UAS-dsRNA numb]-3779r-3 / + Fig. 4a-a shi ts1 / Y ; M[3xP3-RFP.attP.w+.NiGFP]51D / + ; P[neur- Histone2B-RFP] / + Fig. 4e-e N 55e11 w 1118 P[Ubx-flp] P[neur-Histone2B-RFP]619 / Y ; M[3xP3-RFP.attP.w+.NiGFP]51D / + ; FRT82B P[ubinlsGFP] / FRT82B Dl Rev10 Fig. 5c,c N 55e11 w 1118 P[Ubx-flp] P[neur-Histone2B-RFP]619 / Y ; M[3xP3-RFP.attP.w+.NiGFP]51D / + ; FRT82B P[ubinlsGFP] / FRT82B Dl Rev10 spdo G104 Fig. 5d-d w 1118 ; ap-gal4 / + P[UAS-Spdo]G1 / + w 1118 ; neurp-gal4 / P[UAS-SpdoL2-Cherry] Fig. 5e-f Fig. 6a-c
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